This invention relates to an apparatus and method for the classification of white blood cells using two large angle light scattering channels.
Optical flow cytometry is an established means of counting and classifying particles contained within a fluidic sample (Shapiro, H. M., Practical flow cytometry, Third Edition, Wiley-Liss publishers, New York, 1995). One application involves the analysis of a blood sample for the purposes of determining the numbers of platelets, red blood cells (RBCs), and white blood cells (WBCs) per unit volume. This is a common clinical measurement, and optical cytometers have been incorporated into a number of commercial hematology analyzers. Recently, microfluidic techniques have been employed for the purposes of developing cytometers which require smaller sample and reagent volumes (Altendorf, E. et al., xe2x80x9cDifferential blood cell counts obtained using a microchannel based flow cytometer,xe2x80x9d Sensors and Actuators [1997] 1:531-534; Sobek, D. et al., xe2x80x9cMicrofabricated fused silica flow chambers for flow cytometry,xe2x80x9d Solid-State Sensor and Actuator Workshop, Hilton Head Island, S.C. [1994]; Miyake, R. et al., xe2x80x9cA development of micro sheath flow chamber,xe2x80x9d Proceedings of the IEEE micro electro mechanical systems workshop, Nara, Japan [1991] 265-270). Analytical instruments based on these efforts will be smaller and more portable than conventional devices.
Knowledge of the number and nature of blood cells is important for disease diagnosis. For this reason, complete blood counts and white cell differentials are common clinical diagnostic tests carried out using a hematology analyzer. Forward angle light scattering (FALS) is sensitive to particle size and can be used to distinguish platelets from red blood cells. Small angle light scattering (SALS) or large angle light scattering (LALS) in combination with FALS can distinguish within the WBCs between granulocytes, lymphocytes and monocytes (Salzman, G. C. et al. (1975), xe2x80x9cCell Classification by Laser Light Scattering: Identification and Separation of Unstained Leukocytes,xe2x80x9d Acta Cytologica 19:374-377). However, within the granulocytes, SALS and FALS cannot clearly distinguish between eosinophils and the remaining granulocytes such as neutrophils and basophils.
The difference in intensity of scattered light between s-polarized and p-polarized light can be used to further distinguish between the granulocytes. At small angles scattering of the two polarizations is indistinguishable. At large angles, WBC sized structures with no internal structure show only a small difference between the scattered light intensity of the two polarizations. Granulocyte WBCs, having an internal structure comprising numerous small granules, exhibit a difference in scattering intensity between the polarizations. In eosinophils the granules are birefringent and act to depolarize the scattered light, thereby reducing the difference in scattering intensity between the two polarizations. This depolarization has been used to distinguish cell types (Terstappen, L.W.M.M. et al. (1988), xe2x80x9cFour-Parameter White Blood Cell Differential Counting Based on Light Scattering Measurements,xe2x80x9d Cytometry 9:39-43; de Grooth et al., U.S. Pat. No. 5,017,497; Marshall, U.S. Pat. No. 5,510,267. The depolarization was measured by impinging polarized light on a sample, collecting the large angle scattered light at a single large angle, splitting the collected light into two beams and measuring the scattered light in the two beams using two detectors, one for orthogonal light scattering of all polarizations, and the second preceded by a polarizing filter to measure depolarized orthogonal light scattering.
This invention provides an analyzer and a method for distinguishing polarization-preserving particles from depolarizing particles without requiring polarizing filters. This analyzer is especially useful with planar flow cells but can also be used with conventional round flow cytometers. The analyzer comprises a polarized light source positioned to produce a light beam which intersects a liquid sample flow in a flow cell. Light scattered by particles in the sample is measured by a first and a second large angle light scattering channel, positioned to receive scattered light at large angles xcex81 and xcex82, respectively. Each LALS channel comprises a photodetector and collection optics. No polarizing or wavelength filters are required as part of the LALS detection channels. The scattered intensity at each channel is obtained and the relative intensities are used to classify the particles. While this invention can be used with any type of flowing particle, it is particularly suited to a hematology analyzer used to count and classify blood cells, and in particular eosinophils. Preferably xcex81 is between about 15xc2x0 and about 50xc2x0, more preferably about 30xc2x0xc2x110xc2x0, and most preferably about 39xc2x0xc2x110xc2x0 where the flow cell is positioned at an oblique angle to the light beam. Preferably, xcex82 is between about 50xc2x0 and about 130xc2x0, more preferably about 90xc2x0xc2x115xc2x0, or about 115xc2x0xc2x110xc2x0, and most preferably about 73xc2x0xc2x110xc2x0 where the flow cell is positioned at an oblique angle to the light beam.
The flow cell is preferably planar and positioned at Brewster""s angle to the light beam, which for glass and plastic windows in the flow cell is about 56xc2x0. The analyzer can further include small angle and forward angle light scattering channels, which can be used to distinguish between particles based on size and shape. The forward angle light scattering (FALS) detector is preferably placed at an angle xcex8F of between about 0.5 and about 3xc2x0, although it can be placed at a 0xc2x0 angle for absorption measurements. When the FALS detector is used for absorbance measurements with a laser light source, filters will be required as is known to the art because of the intensity of the light beam. The small angle light scattering detector (SALS) is preferably placed at an angle xcex8S greater than xcex8F, of between about 2xc2x0 and about 10xc2x0. The analyzer can further include an absorption channel for measuring particles containing absorptive species such as dyes, hemoglobin and bilirubin. The absorption measurement can be carried out in the same stream as the scattering measurement, in the same or a different measurement zone, or in a separate stream. The absorption channel comprises a light source positioned to illuminate a sample flow, collection optics positioned to collect the transmitted light, and a photodetector. The collection optics can include a wavelength filter. The analyzer can further include additional detectors such as a second absorption channel comprising collection optics with a second wavelength filter and a photodetector. The analyzer can further include a fluorescence channel, comprising a fluorescence photodetector and fluorescence collection optics. The fluorescence channel can utilize the scattering light source or a separate light source.
This invention also provides a method for distinguishing polarization preserving particles from depolarizing particles, comprising the steps of: flowing said particles through a p-polarized light beam; measuring the scattered light intensity I(xcex81) at a first large angle, xcex81; measuring the scattered light intensity I(xcex82) at a second large angle, xcex82, wherein xcex82 greater than xcex81; and comparing I(xcex81) to I(xcex82), thereby distinguishing polarization preserving particles from depolarizing particles.